Frequently in chemical, biological or materials assay procedures it is necessary to isolate or purify a desired substance/s from other substance/s. Column chromatography of various types are commonly used for such procedures. In particular, a simple form of chromatography, namely spin-column chromatography is frequently employed to enrich for analytes, to reduce or remove interferences or combinations of these purposes. Such spin-columns are of simple design and usually contain a separation media such as cross-linked dextrans or polyacrylamides, ion-exchange resins or silica gels of various types.
Generally, the spin-column devices are prepared by having the separation media suspended in a buffer or organic solvent within the spin-column. Before centrifugation the separation media has an essentially uniform distribution in the spin column and when viewed perpendicular to its surface has a circular cross section. Usually, the spin-column is pre-spun in a centrifuge, preferably equipped with a “swinging-bucket” rotor to keep the bulk mass of the separation media within the column from slanting and to maintain a uniform flow path for the suspension media and sample material that is parallel to the walls of the column. Following centrifugation in a “swinging bucket” rotor the separation media has an essentially uniform distribution in the spin column and when viewed perpendicular to its surface has a circular cross section. A fixed angle rotor is sometimes used, but the column media shifts and forms an angled surface. Following centrifugation in a “fixed angle” rotor the separation media has an uneven distribution in the spin column and when viewed perpendicular to its surface has an elliptical or ovoid cross section. Applied samples then flow at an angle through the separation media and not parallel to the column walls.
Regardless of the type of rotor used, the applied sample must preferably be loaded to the center of the media. Frequently, the spin-column is pre-spun to force the liquid suspending the separation media through the column to remove excess liquid from the spin-column and compact the separation media. The sample is then carefully loaded onto the top center of the separation media in the spin-column and the spin-column is centrifuged in a collection tube and the desired material is collected in the collection tube. When spin-columns have been pre-spun, care must be taken that the spin-column is in the identical orientation as it was during the pre-spin, otherwise the media and applied sample will be redistributed during centrifugation and the desired separation/filtration will be inadequate or compromised.
In some cases, the desired material is retained on the separation media within the spin-column and is subsequently eluted, sometimes after washes to remove undesired materials, with the desired material being displaced from the separation media within the spin-column by some alteration of conditions facilitating its release from the column separation media, and sometimes the desired material is not bound and interferences are bound to the separation media.
There are many examples of the use of spin-columns, for example to accomplish buffer exchange of proteins, such as antibodies, or to separate unincorporated radioactive nucleotides from polynucleotides which have incorporated the radiolabel, for example, following radioactive phosphorylation of DNA for use as probes from γ-32P-ATP. There are many other examples well known in the art and described in many manuals or web sites describing molecular biology, labeling or purification protocols or other techniques. See for example, CSH Protocols at Cold Spring Harbor Protocols Online or the Wikipedia location on the world wide web, entitled “Spin-column-based nucleic acid purification,” for a description of minicolumn DNA preparation.
Other useful guides are: Protein analysis and purification: benchtop techniques, by Ian M Rosenberg, Birkhäuser, Boston©2005, 2nd ed pp 337-339, which describes use of spin columns to accomplish buffer exchange; Affinity Chromatography: Methods and Protocols, by Michael Zachariou, Springer, New York, 2007, pp. 156-157, which describes affinity purification from cell lysates using spin columns by centrifuge and by vacuum; Hoyt P R, Doktycz M J, Warmack R J, Allison D P., Spin-column isolation of DNA-protein interactions from complex protein mixtures for AFM imaging., Ultramicroscopy. 2001 January; 86(1-2):139-43; and Nickoloff J A, Sepharose spin column chromatography. A fast, nontoxic replacement for phenol:chloroform extraction/ethanol precipitation. Mol Biotechnol. 1994 February; 1(1):105-8.
Often, to obtain proper separation, the spin-column devices must be centrifuged in a swinging bucket type of rotor to keep the media from slanting and distorting the separation process. When spun in a fixed angle rotor the separation media becomes distorted or becomes slanted and the applied sample material and liquids experience various separation path lengths or channeling through the column, with the sample material in the lower aspect of the slanted media experiencing a short separation path and the sample material at the high end of the slope experiencing longer separation paths. This behavior of the applied sample material is in conflict with good separation performance. Ideally, as is well known in chromatography theory and practice, the separation media should have similar effective separation paths across the diameter of the separation media and along the length or depth of the separation media; however, channeling or significant distortions of the separation path greatly compromises and significantly alters and adversely affects the performance of the separation process. There is therefore a need for more convenient, efficient and effective spin column devices that overcome the problems of channeling, special centrifuge rotors, centered sample loading, and single purpose utility of present spin column devices.